Common carp (Cyprinus carpio) CD81 promoting CyHV-3 virus replication via regulating autophagy and RLRs-interferon signaling pathway.

Cyprinid herpesvirus type 3 (CyHV-3), also called Koi herpesvirus (KHV), which leads to mass cyprinid mortality and enormous economic losses. To establish an infection, CyHV-3 needs to counteract host antiviral responses. CD81 belongs to the evolutionary conserved tetraspanin family of proteins. Several studies have shown that different members of the tetraspanin superfamily modulates different virus infectious processes. Here we aimed at analysing the role of CD81 in CyHV-3 infection. In this study, we cloned and characterized the CD81 of Common Carp, the open reading frame of CcCD81 gene was 702 bp, which encoded 234 amino acids with four transmembrane domains (TM1 to TM4), a small extracellular loop (SEL), and a large extracellular loop (LEL). Tissue distribution analysis showed that CcCD81 was widely expressed in all the tested tissues with the highest expression in head kidney, followed by a high expression in brain. Subsequently, expression levels of CcCD81 were significantly increased in CCB cells within the first 3h after infection, meanwhile, the expression of viral gene VP136 was reduced after CcCD81 knockdown in CCB cells post CyHV-3 infection. Furthermore, CcCD81 knockdown can significantly reduce the autophagy process and increase the promoter activity of ISRE and IFN-1 in the CCB cells after viral infection, as well as other genes involved in the IFN signaling pathway, including RIG-1、MDA5、MAVS、TBK1 and IRF3. Taking the data together, we revealed that CcCD81 mediates autophagy and blocks RIG-1-mediated antiviral signaling and negatively regulates the promoter activity of type I interferon (IFN) promoting virus replication. These results reveal a new link between autophagy and four-transmembrane-domain protein superfamily and contribute to elucidate the mechanism of CyHV-3 infection.

Autophagy induced by Cyprinid herpesvirus 3 (CyHV-3) facilitated intracellular viral replication and extracellular viral yields in common carp brain cells.

Autophagy is a conservative and important process that exists in all eukaryotic cells in nature. Cyprinid herpesvirus 3 (CyHV-3), also known as KHV (Koi Herpesvirus), is a pathogen that mainly infecting common carp and koi. In the present study, we identified the CcLC3B gene, with a length of 379 bp and displaying a close evolutionary relationship with other sixteen different species, the tissue distribution and expression pattern of CcLC3 were also identified. We found that CyHV-3 infection could promote autophagy in CCB cells at the early stage but inhibit autophagy at the late stage by using confocal fluorescence microscopy, transmission electron microscopy and western blotting. And we measured the protein levels associated with the Akt/mTOR signalling pathway, intracellular replication of CyHV-3 at the mRNA and protein levels as well as viral titters. Collectively, the results taken together suggested that CyHV-3 infection could promote autophagy in CCB cells at the early stage but inhibit autophagy at the late stage via mTOR and that promoting autophagy could facilitate CyHV-3 intracellular replication and extracellular viral yields in CCB cells. These findings revealed the relationship between CyHV-3 and autophagy and provided a novel treatment strategy targeting the autophagy signalling pathway against CyHV-3 infection.

Characterization of the Complete Mitochondrial Genome of the Spotted Catfish Arius maculatus (Thunberg, 1792) and Its Phylogenetic Implications.

The spotted catfish, Arius maculatus (Siluriformes), is an important economical aquaculture species inhabiting the Indian Ocean, as well as the western Pacific Ocean. The bioinformatics data in previous studies about the phylogenetic reconstruction of Siluriformes were insufficient and incomplete. In the present study, we presented a newly sequenced A. maculatus mitochondrial genome (mtDNA). The A. maculatus mtDNA was 16,710 bp in length and contained two ribosomal RNA (rRNA) genes, thirteen protein-coding genes (PCGs), twenty-two transfer RNA (tRNA) genes, and one D-loop region. The composition and order of these above genes were similar to those found in most other vertebrates. The relative synonymous codon usage (RSCU) of the 13 PCGs in A. maculatus mtDNA was consistent with that of PCGs in other published Siluriformes mtDNA. Furthermore, the average non-synonymous/synonymous mutation ratio (Ka/Ks) analysis, based on the 13 PCGs of the four Ariidae species, showed a strong purifying selection. Additionally, phylogenetic analysis, according to 13 concatenated PCG nucleotide and amino acid datasets, showed that A. maculatus and Netuma thalassina (Netuma), Occidentarius platypogon (Occidentarius), and Bagre panamensis (Bagre) were clustered as sister clade. The complete mtDNA of A. maculatus provides a helpful dataset for research on the population structure and genetic diversity of Ariidae species.

Genome-Wide Analysis of Differentially Expressed mRNAs and lncRNAs in Koi Carp Infected with Koi Herpesvirus.

Long noncoding RNAs (lncRNAs) constitute an emerging group of ncRNAs that modulate gene expression at the transcriptional or translational level. Koi herpesvirus (KHV), also known as Cyprinus herpesvirus type 3 (CyHV-3) and characterized by high pathogenicity and high mortality, has caused substantial economic losses in the common carp and koi carp fisheries industry. In this work, we sequenced the lncRNA and mRNA of host koi carp infected with KHV. A total of 20,178 DEmRNAs were obtained, of which 5021 mRNAs were upregulated and 15,157 mRNAs were downregulated. Both KEGG pathways and GO terms were enriched in many important immune pathways. The KEGG analysis showed that DEGs were significantly enriched in many important immune pathways, such as apoptosis, NOD-like receptor signaling pathway, Jak-STAT signaling pathway, TNF signaling pathway, IL-17 signaling pathway, NF-kappa B signaling pathway, and so on. Furthermore, a total of 32,697 novel lncRNA transcripts were obtained from koi carp immune tissues; 9459 of these genes were differentially expressed. Through antisense, cis-acting, and trans-acting analyses, the target genes of differentially expressed lncRNAs (DElncRNAs) were predicted. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the DElncRNA expression pattern was consistent with the differential mRNA expression pattern. The lncRNA-mRNA network analysis, which included many immune pathways, showed that after KHV infection, the expression of most lncRNAs and their target mRNAs were downregulated, suggesting that these lncRNAs engage in a positive regulatory relationship with their target mRNAs. Considering that many studies have shown that herpesviruses can escape the immune system by negatively regulating these immune pathways, we speculated that these lncRNAs play a significant role in KHV's escape from host immunity. Furthermore, 10 immune-related genes and 20 lncRNAs were subsequently verified through RT-qPCR, to confirm the accuracy of the high-throughput sequencing results. In this study, we aimed to explore lncRNA functions in the immune response of lower vertebrates and provide a theoretical basis for the study of noncoding RNAs in teleosts. Therefore, exploring lncRNA expression in KHV-infected koi carp helped us better understand the biological role played by lncRNA-dependent pathways in aquaculture animal viral infection.

Identification and expression analysis of cellular and viral microRNAs in CyHV3-infected KCF-1 cells.

MicroRNAs (miRNAs) are small non-coding RNAs with approximately 22 nucleotides (nt) that are encoded by a diverse range of metazoan eukaryotes, plants and viruses. CyHV-3 (cyprinid herpesvirus-3) is a member of the Alloherpesviridae virus family and has caused severe economic losses for the common carp and koi carp fishery industries. In this study, a total of 15,987,652 clean reads were generated from a cDNA library of CyHV-3-infected KCF-1 (koi caudal fin) cells using high-throughput sequencing technology. Following annotation and secondary structure prediction, 28 miRNAs were identified as novel candidate miRNAs encoded by common carp (Cyprinus carpio), and seven miRNAs were shown to be encoded by CyHV-3. Next, 19 host miRNAs and seven viral miRNAs were validated by stem-loop real-time PCR. Northern blot analysis confirmed the presence of 14 host miRNAs and five CyHV-3-encoded novel miRNAs. The results of this study expand the knowledge of common carp and CyHV-3 microRNAs and provide a useful theoretical foundation for further study of CyHV-3.

Transcriptomic analysis of koi (Cyprinus carpio) spleen tissue upon cyprinid herpesvirus 3 (CyHV3) infection using next generation sequencing.

Cyprinid Herpesvirus 3 (CyHV-3) can infect and specifically cause a huge economic loss in both common carp (Cyprinus carpio) and its ornamental koi variety. The molecular mechanisms underlying CyHV-3 infection are not well understood. In this study, koi spleen tissues of both mock and CyHV-3 infection groups were collected, and high-throughput sequencing technology was used to analyze the differentially expressed genes (DEGs) at the transcriptome level. A total of 105,356,188 clean reads from two libraries were obtained. After the de novo assembly of the transcripts, 129,314 unigenes were generated. Of these unigenes, 70,655 unigenes were matched to the known proteins in the database, while 2190 unigenes were predicted by ESTScan software. Comparing the infection group to the mock group, a total of 23,029 significantly differentially expressed unigenes were identified, including 10,493 up-regulated DEGs and 12,536 down-regulated DEGs. GO (Gene Ontology) annotation and functional enrichment analysis indicated that all of the DEGs were annotated into GO terms in three main GO categories: biological process, cellular component and molecular function. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis of the DEGs showed that a total of 12,002 DEG unigenes were annotated into 256 pathways classified into 6 main categories. Additionally, 20 differentially expressed genes were validated by quantitative real-time PCR. As the first report of a transcriptome analysis of koi carp with CyHV-3 infection, the data presented here provide knowledge of the innate immune response against CyHV-3 in koi carp and useful data for further research of the molecular mechanism of CyHV-3 infection.

Whole-genome sequence of a novel Chinese cyprinid herpesvirus 3 isolate reveals the existence of a distinct European genotype in East Asia.

Cyprinid herpesvirus 3 (CyHV3), also known as koi herpesvirus (KHV), can be subdivided primarily into European and Asian genotypes, which are represented by CyHV3-U or CyHV3-I and CyHV3-J, respectively. In this study, the whole genome sequence of a novel Chinese CyHV3 isolate (GZ11) was determined and annotated. CyHV3-GZ11 genome was found to contain 295,119 nucleotides with 52.9% G/C content, which is highly similar to those of published CyHV3-U, CyHV3-I, and CyHV3-J strains. With reference to CyHV3-U, CyHV3-I, and CyHV3-J, CyHV3-GZ11 was also classified into 164 open reading frames (ORF), which include eight repeated ORFs. On the basis of the 12 alloherpeviruses core genes, results from phylogenetic analysis showed that CyHV3-GZ11 had closer evolutionary relationships with CyHV3-U and CyHV3-I than with CyHV3/KHV-J, which were also supported by genome wide-based single nucleotide substitution analysis and the use of a series of developed molecular markers. This study was the first to reveal the presence of a distinct European CyHV3 genotype in East and Southeast Asia at a whole genome level, which will evoke new insights on exploring the origin, evolution, and epidemiology of the virus.

Extracellular virion proteins of two Chinese CyHV-3/KHV isolates, and identification of two novel envelope proteins.

Proteins in extracellular virions of two Chinese cyprinid herpesvirus-3/koi herpesvirus (CyHV-3/KHV) isolates were identified through one-dimensional gel-based matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry in combination with liquid chromatography tandem mass spectrometry (LC ESI-MS/MS). A total of 43 viral proteins were identified, seven of which (pORF62, 68, 43, 51, 92, 84, and 72) were characterized as marked characteristic viral proteins in CyHV-3/KHV virions and six of which (pORF11, 27, 83, 91, 106 and 116) were not detected previously. Of the newly identified proteins, pORF83 was identified as a novel viral minor envelope protein by Western blot analysis and indirect immunofluorescence assay. Another 27 cellular proteins were validated by LC ESI-MS/MS to be involved in purified extracellular CyHV-3/KHV virions, among which a highly abundant, virus-inducible stress protein was shown and characterized as a cell-derived envelope protein in CyHV-3/KHV virions. Our study extends the number of known CyHV-3/KHV virion-associated viral proteins from 40 to 46 and identifies a novel viral envelope protein as well as a cellular envelope protein. The present findings provide some new insights for better understanding CyHV-3/KHV.